The hormonal induction of rat hepatic zinc metallothionein (Zn MT) will be studied in vivo and in vitro (isolated, cultured rat hepatocytes and human hepatoma HepG2 cells). Isoproterenol (beta-agonist) and 2-chloroadenosine (purinergic agonist) will be used to activate the cAMP-dependent protein kinase (protein kinase A) pathway. Norepinephrine (alpha-agonist) phorbol ester (TPA), and calcium ionophore (A23187). Zn MT mRNA and protein levels will be determined during inductions, using a dot blot assay with a 21-mer probe and a [109Cd]-zinc displacement assay, respectively. The importance of the protein kinase B pathway for induction of Zn MT will be studied. Attempts will be made to identify protein kinase B-responsive elements in the MT promoter and to isolate protein kinase B cascade transcription factors, as has been done for AP1, AP2, and Spl. DNAse I footprinting assays, CAT assays and transfections, oligonucleotide-affinity chromatography, and gel shift assays will be learned by this lab and used in these studies. The MT promoter and hormonal induction on Zn MT are fruitful and physiologically interesting systems to study in order to elucidate molecular events occurring in eucaryotic hormonal signalling cascades from the plasma membrane to a gene in the nucleus.